Endotoxin Detection by LAL for Parenteral Products

Endotoxin Detection by LAL for Parenteral Products

byHongjin Min

CR Endosafe Asia Technical ManagerWoo Jung BSC, Inc.

August 25, 2003 Woo Jung BSC, Inc. 2

Principal Microbiology Tests

STERILITY: Detection of live microorganisms

PYROGEN: Detection of cell-wall fragments from Gram(-) bacteria. History of a product’s exposure to GNB

Charles River Endosafe


Endotoxins and Pyrogens

Pyrogens are fever-inducing agents in humans and animals

include endotoxin, gram + cell debris, fungi

Endotoxins are components from the outer membrane of gram-negative bacteria





EndotoxinEndotoxin - Component from outer membrane of gram-negative bacteria

Environmental (unpurified) endotoxin: complex of protein, carbohydrate & lipid Purified endotoxin: lipopolysaccharide molecule of

polysaccharide and lipid a, without protein



Purified Endotoxin -LipopolysaccharidePasses through sterilizing membrane filters

Lipid A region causes LAL reaction and potent pharmacological activities associated with endotoxin

poorly dispersible in water

pyrogenicity correlates with LAL assays



Why Test for Endotoxins?

To protect against adverse reactions (sepsis) in humans and animals

FDA and USP guidelines require final product testing on all parenterals and medical devices

To safeguard against diminished effectiveness of a product due to endotoxin



Endotoxicity

ENDOTOXIN CAUSES MYELOID TISSUE TO RELEASE INFLAMMATORY MEDIATORS

CYTOKINES INDUCE A VARIETY OF TISSUE DAMAGE

SHOCK and MULTIPLE ORGAN DYSFUNCTION MAY OCCUR



How to test for Endotoxins?

Rabbit Pyrogen TestFD approved in 1941

LAL / TAL Testgel clot LAL approved in 1973kinetic LAL in 1987

Cell based assaysIn Vitro pyrogen assay



Rabbit Pyrogen Test

Measure the change in

temperature of 3 rabbits,

after intravenous

injection

of the test solution.



LALLimulus - genus of Horseshoe Crab

Amoebocyte - blood cells

Lysate - blood extract

LAL is a FDA regulated biological that reacts chemically in the presence of gram negative cell debris. This reaction can be measured and quantified.






LAL Discoveries by Bang and Levin

Described role of endotoxin in coagulation of Limulus blood

Prepared Endotoxin - responsive lysate from Amoebocytes



LAL Assay

LAL is > 100 times more sensitive, specific and accurate

Economy of LAL permits more testing

Tolerance of rabbits to endotoxin is a concern for FDA

LAL activity correlates with pyrogenicity



History of LAL Regulations

1941 Rabbit Pyrogen test included in USP

1973 FDA announced LAL as a biological product

1977 FDA described conditions for use of LAL as end-product test for human biological & medical devices

1980 Federal register drafted guidelines for use of LAL test for end-product testing of human & animal injectable drug products



History of LAL Regulations1983 Federal Register lists final guidelines on

LAL testing including chrom and turb

1987 USFDA LAL - Test Guideline

1991 Interim Guidance - Kinetic LAL Test

1995 USP 23 Bacterial Endotoxin Test

1998 European Pharmacopoeia BET

2000 Harmonized BET



Other Regulatory Documents

USP monographs

Manufacturer’s Package Insert

Internal SOP

AAMI guidelines

other Pharmacopoeia



New Harmonized BET

Effective July 2000

First Microbiological test harmonized by ICH

Allows use of endotoxin-specific LALmust test for glucans if suspected in your sample

Includes kinetic LAL methods


Gel Clot & Kinetics –The Basics



Clotting Reaction For Activated LAL

Endotoxin - Glucan

Pro-ClottingEnzymes

ClottingEnzyme Substrate

Coagulogen Coagulin GelM++ pH=7.2



LAL TestingGel Clot(Limit Test)Gel Clot(Semi-Quantitative)Endpoint ChromogenicEndpoint TurbidimetricKinetic ChromogenicKinetic Turbidimetric



Summary of Gel Clot Test

Endpoint sought by 180 inversion of sample tube

Positive = Firm GelNegative = Flow down side of tube




Gel Clot Reagent

In generalLowest reagent cost

Simple reporting (+-)

Found in all

Pharmacopoeia

Good stability

Simple technique

Endosafe

firm gel

buffered lysate

better interference resistance

.015 EU/ml



Sources of gel clot test problemsUse of wrong EU/ng ratio

use of improper reaction or dilution tubes

Incubating temperature too hot or too cold

Improper dilutions

Improper handling of lysate



Gel Clot Disadvantages

Limits test

Least sensitive

Most time consuming

Poor trending options

Variability, subjectivity



KINETIC LAL TESTING

ENDOTOXIN

CLOTTING CASCADE

CLOT

coagulogen

proclotting enzyme

chromogenic substrate pNA

clotting enzyme

coagulin





Microplate Absorbance Readers



Kinetic LAL Advantages

Quantitative results

Higher throughput (96 wells)

Lower sensitivity (to .001)

Wider standard curve range

Clear concise reporting

Allows for data trending





MEASURE CHANGES IN OPTICAL DENSITY (OD)

RELATES CHANGES IN OD TOENDOTOXIN CONCENTRATION

AUTOMATED CALCULATIONS

REPORTS RESULTS of ANALYSIS

FUNCTION: Kinetic LAL SystemCharles River Endosafe


Reaction Time

Optical Density

Principles of Quantitation

Change in O.D. over Time


Optical Density

Time

Quantitative LAL Test Results

Endpoint: “Fixed Time”


OD

Endotoxin Conc.

Standard Curve:

Endpoint TestBeer’s Law: Absorb. vs. Conc.

positive slope


Optical Density

Reaction Time

Quantitative LAL Results“Fixed” Optical Density


Standard Curve:

Kinetic Test

log (Endotoxin Conc.)

Log(ReactionTime)

negative slope


Optical Signals from LPS Interaction with Kinetic LA

Reaction Time

OD 50 5 0.5 0.05 0.005

NC

Onset OD323 499 801 1379 2401


Optical Signals from LPS Interaction with Kinetic LAL

Reaction Time

OD 50 5 0.5 0.05 0.005

NC

0.2

0.05

0.1282 440 700 1161 1837

323 499 801 1379 2401

368 565 914 1643 3262

R

-0.995

-0.999

-1.000


How to Quantify the Amount of Endotoxin in the Sample

A standard curve is constructed by plotting

the log of onset time

versus

the log of concentration Log ( Endotoxin Conc. )

Log

( Rea

ctio

n Ti

me

)

Sample



Summary of Kinetic AssayThe amount of endotoxin present is inversely proportional to the time of reaction

the more endotoxin present, the quicker the well or tube will react (change color or become turbid)

A standard curve is generated based on how long each of the standards take to reach an onset OD

Unknowns are then compared to the standard curve to generate a endotoxin values


Selecting Kinetic LAL Reagents

Turbidimetric or Chromogenic?



Selection of Kinetic LAL Reagent

cost of reagent

unique interference problems

analysis time

sensitivity



Kinetic Lysate Options

Turbidimetricleast expensive, 2-log range down to .01

KTA2

mid-priced, 3-log range down to .005, better interference resistance

Chromogenicmost expensive, 4-log range (50-.005), best interference resistance



Kinetic Chromogenic ReagentIn general

4 log standard curve

Low sensitivity (.005)

Fast reaction

Best for biologicals

Most expensive

lysate

EndosafeFastest (.005 in 50

min)

Less expensive

Flexible packaging

Best linearity

Best stability



KINETIC CHROMOGENIC (KCA)ENDOCHROME-K, LAL

REHYDRATE VIAL WITH 3.2 - 3.4 mL LRW

SET WAVE LENGTH of READER to 405 nm.

SET ONSET OD to 0.1 OD UNITS

SET UP 2 to 4 LOG RANGE

EXPECT INCUBATION TIME < 40 MINUTES



Kinetic Turbidimetric Reagent

In GeneralPrice similar to gel

Linear over two logs

Stable

.05 in about 50

minutes

Trend analysis

Endosafe KTALinear over wider rangeBetter spike recoveryproducts other than waterAlso true gel clotNo reconstitution buffer



KINETIC TURBIDIMETRIC (KTA)ENDOSAFE KTA LAL

REHYDRATE VIAL WITH 5.2 mL OF LRW

SET WAVE LENGTH OF READER TO 340 nm.

SET ONSET OD to 0.05 OD units

SET UP 2 or 3-LOG RANGE

EXPECT INCUBATION TIME <45 MINUTES



KTA2 Turbidimetric Lysate

1st turb lysate to run at .005 EU/ml

Faster than existing turbs (.005 in < 1 hour)

Interference resistance of chromogenic lysate

Priced closer to conventional turb

Can be used with polynomial regression



Glucans ((1-3)-B-D-glucan)

Materials that cause false positive in LAL by activating Factor-G pathway

Non-pyrogenic

Sources include cellulosic membranes and fungal or yeast extract

Reacts like endotoxin in gel clot

Causes enhancement in kinetic methods



LAL and Glucans

No current LAL on the market is totally

unreactive to glucans

Reactivity tied to extraction method

Variability between lots

Best way to test today is to use glucan

blocker



LAL - Test Interference

Inhibition:Recovery < Expected Amount

Enhancement:Recovery > Expected Amount

Non-interference:

Recovery = Expected Amount



Calculate EL, MVD, MVCMake sample dilutions between undiluted and MVDScreen these dilutions to determine non-interfering test concentrationOnce a non-interfering sample dilution is determined perform validation on three lots of this product

Product Screen (Inh / Enh)



Measure and document pH of sample + LAL

If no pH adjustment is made then, the validation data serves as documentation for routine testing, and pH measurements will not have to be performed routinely

If pH adjustments are made then routine testing should include pH measurement and documentation.

Product Screen (Inh / Enh)



LAL CompatibilityA test sample is compatible with LAL reagent if the positive product control (ppc) is recovered.

in gel-clot, at least 2-lambda is recovered

in kinetic LAL, 100 50% of a known amount of CSE is recovered relative to a standard curve.



Scope of Interference Problem

Only 25% of pharmaceuticals are testable undiluted

Inhibition is usually solved by dilution

Devices are seldom inhibitory, unless coated



Eliminating Causes Of Interference

PROBLEM

pH

Endotoxin loss

Mg depletion

LAL-RM

SOLUTION

Buffered LAL Reagent

In-plate spiking

Permissible Dilution

Endotoxin-Specific buffer



Current Trends in LAL Testing

Continued move toward kinetic testingover 55% of all LAL tests performed today

Compliant software

Database trending

Robotics

In-vitro pyrogen test


LAL Applications



LAL Applications

Pharmaceuticals

Medical Devices

Water testing

Dialysis testing

Cell culture



Acceptance of LAL by Pharmaceutical Industry

LAL IS > 100 TIMES MORE SENSITIVE, SPECIFIC and ACCURATE

ECONOMY of LAL PERMITS MORE TESTING

TOLERANCE of RABBITS to ENDOTOXIN = CONCERN for FDA



Baxter Report on Non-Endotoxin Pyrogens

143,196 LAL and 28,410 RABBIT TESTS

ALL PYROGENIC TESTS GAVE POSITIVE LAL RESULT

NO FALSE-POSITIVE LAL TESTS

NOT ALL POSITIVE LAL TESTS WERE PYROGENIC in RABBITS

RABBIT WAS OFTEN UNRELIABLE



Improved Quality of U. S. Pharmaceuticals1974 FDA STUDY FOUND 45% of ALBUMIN PYROGENIC

REGULATED In-PROCESS TESTING ELIMINATED PYROGENIC ALBUMIN

PYROGENS REMOVED from INFLUENZA VACCINES

LAL REQUIRED for SPINAL-FLUID DRUGS

NO OUTBREAKS of FEVER SINCE 1980



LAL Application - Medical Devices

1987 FDA Guideline

Extraction test

Endotoxin Limit -0.05 EU/ml up to 20 EU/device

Turbidimetric most cost effective method for testing



Medical Devices Requiring Pyrogen(Endotoxin) Testing

DEVICES that PENETRATE SKIN

MEDICAL IMPLANTS

FLUID PATHWAYS for IV Medications

NOT APPLICABLE to:GLOVES, ORTHOPEDIC PRODUCTS,WOUND DRESSINGS



Official Role for LAL Testing in Device Quality

Comparison Study revealed LAL advantages over Pyrogen (rabbit) Test

DEVICE INDUSTRY SWITCHED TO LAL TESTING BY 1987

DEVICES with NON-PYROGENIC LABEL REQUIRE LAL TEST



LAL Application - Water system

Routine monitoring

Turbidimetric is the lysate of choice

Trending allows you to set action and alert limits



Sources of Endotoxin in Water System

COLONIZATION OF SOFTENERS,CARBON BEDS and DI UNITS by G(-) RODS

BIOFILM on RO MEMBRANES, WATER PIPES, and in STORAGE TANKS



LAL Application - Dialysis

Testing of dialysis water and concentrate

Testing performed in clinics, hospitals, and reference labs

Gel clot is method of choice because of low volume of tests

New AMMI limit on water for dialysis is 2 EU/ml



LAL Application - cell culture

Effects of endotoxin very greatly depending on cell line.

in vitro fertilization adversely affected

Classified broadly as a parenteral productEndotoxin testing by BET is imminent
